The DNA damage response (DDR) ensures genomic stability and survival under genotoxic stress conditions. One of the DDR effector pathways is homologous recombination, a DNA repair option that addresses DNA double-stranded breaks and other complex DNA damage such as interstrand crosslinks. Induction of such types of DNA damage constitutes the functional principle of a large number of cancer treatment modalities, highlighting the importance to understand the underlying biological processes. A long-term goal is to identify the mechanism of recombinational DNA repair and how they are regulated by the DDR. Specifically, we focus on the mechanism and regulation of the assembly of the Rad51-ssDNA filament, a key intermediate in recombinational repair. Another goal is to identify negative and positive control mechanisms that govern DDR signaling and how they affect cell cycle phase-specific regulation. Defects in the DDR and recombinational repair predispose individuals to cancer, providing significant impetus to understand the basic biological mechanisms involved. The Specific Aims are: (1) Determine the mechanisms of Rad51-ssDNA filament assembly. The presynaptic Rad51-ssDNA filament exists in a meta-stable balance between its assembly and disassembly. We will test mechanistic models, how the Rad51 paralogs Rad55-Rad57 antagonize the anti-recombination function of Srs2. (2) Regulation of Rad51-ssDNA filament assembly by post-translational modification. Genetic experiments are leading biochemical approaches to examine the effect of post-translational modifications of Rad55-Rad57, Srs2 and PCNA on Rad51-ssDNA filament assembly/disassembly. Genetic and biochemical approaches will identify modification-specific interaction partners of Rad55-Rad57. (3) Determine mechanisms of the G1 DNA damage response. The G1-S border is a major regulatory transition in the mammalian DNA damage response but understudied in yeast. We will identify mechanisms in G1 checkpoint control involving negative control on Mec1 kinase in haploid and diploid yeast cells. (4) Determine the mechanism of positive feedback regulation of Mec1 kinase. While the initial activation of Mec1 kinase is understood to some detail, the mechanisms involved in the maintenance of its activated state remain unclear. We will combine biochemical and genetic approaches to determine the mechanism of positive feedback regulation on Mec1 kinase.